Hantavirus Pulmonary Syndrome Real Time RT
Contents
1. Liferiver Revision No ZJO007 Issue Date Jul 1 2012 Hantavirus Pulmonary Syndrome Real Time RT PCR Kit C c User Manual 20 C Z y For In Vitro Diagnostic Use Only QR 0090 01 aal Shanghai ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 21 34680596 For use with LightC ycler1 0 2 0 Instrument trade liferiver com cn Fax 86 21 34680595 pec rer Obelis S A Boulevard G n ral Wahis 53 1030 Brussels BELGIUM Tel 32 2 732 59 54 2 floor No 15 Building No 188 Xinjunhuan Road Fax 32 2 732 60 03 PuJiang Hi tech Park Shanghai China E Mail mail obelis net 1 Intended Use Hantavirus pulmonary syndrome real time RT PCR kit is used for the detection of Hantavirus pulmonary syndrome HPS in serum by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluorescence signal is detected initially is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating product with2. a 38 T Positive 38 40 25 35 Re test If it is still 38 40 report as 1 PCR Inhibition No diagnosis can be concluded For further questions or problems please contact our technical support at trade liferiver com cn
3. c gene fragments by polymerase chain reaction Fluorescence is emitted and measured by the real time systems optical unit The detection of amplified Hanta virus HPS DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1 In addition the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control IC 4 Kit Contents HPS Super Mix 1 vial 350u1 RT PCR Enzyme Mix 1 vial 28ul Molecular Grade Water 1 vial 400u1 Internal control IC 1 vial 30u1 HPS Positive control 1x10 copies ml 1 vial 30u1 Analysis sensitivity 1 X 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the RNA extraction kits recommended the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Rea
4. er is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge 2 Pipet 15ul Master Mix with micropipets in sterile filter tips to each real time PCR reaction plate tubes Separately add 5ul RNA sample positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 45 C for 10min Icycle 95 C for 15min Icycle 95 C for 5sec 60 C for 30sec Fluorescence measured at 60 C 10 Threshold setting Choose Arithmetic as back ground and none as Noise Band method then adjust the Noise band just above the maximum level of molecular grade water and adjust the threshold just under the minimum of the positive control Selection of fluorescence channels Target Nucleic Acid C 40cycles 11 Quality control Negative control positive control and internal control must be performed correctly otherwise the sample results is invalid Crossing point value 530nm Molecular Grade Water lank 25 35 Positive Control qualitative assay lt 35 gt 12 Data Analysis and Interpretation The following sample results are possible Crossing point value Result Anal Lj 530nm 560nm iene cd 25 35 Below the detection limit or negative
5. l time PCR system e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g e Vortex mixer e RNA extraction kit e Real time PCR reaction tubes plates e Cryo container e Pipets 0 5 ul 1000 ul e Sterile filter tips for micro pipets e Sterile microtubes e Disposable gloves powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks 7 Warnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink and smoke in laboratory e Avoid aeroso
6. ls 8 Sample Collection Storage and Transport e Collected samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transport of etiologic agents 9 Procedure 9 1 RNA Extraction Different brands of RNA extraction kits are available You may use your own extraction systems or the commercial kit based on the yield For RNA extraction kit please comply with manufacturer s instructions The recommended extraction kit is as follows Nucleic Acid Isolation Kit Cat Number RNA Isolation Kit ME 0010 ME 0012 ZJ Biotech QIAamp Viral RNA Mini Extraction Kit 50 52904 QIAGEN 9 2 Internal Control It is necessary to add internal control IC in the reaction mix Internal control IC allows the user to determine and control the possibility of PCR inhibition Add the internal control IC 1ul rxn and the result will be shown in the 560nm Channel 9 3 RT PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 13l 1 yl Tul Super Mix Enzyme Mix Internal Control Spl 15l Extraction RNA Master Mix Reaction Plate Tube PCR Instrument XPCR system without 560nm channel may be treated with lu Molecular Grade Water instead of 11 IC 1 The volumes of Super Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls and sample prepared Molecular Grade Wat
7. out having to re open the reaction tube after the amplification 3 Product Description Hantaviral diseases in humans are caused by a group of closely related trisegmented negative sense RNA viruses of the genus Hantaanvirus of the family Bunyavirididae The type and severity of the disease depends largely on the serotype of the virus involved Two classes of hantavirus associated illnesses have been described HFRS for the disease in which the kidneys are primarily involved and HPS for the disease in which the lungs are primarily affected The recent data concerning the pathogenesis of Hantavirus infection e g Sherif R Zaki confirm that the basic necroinflammatory changes in the infection develop in the blood vessels The endothelium is the internal cover of the blood vessels and the largest endocrine and metabolic organ of the human body Its role is to maintain the balance which has the fundamental importance in the life Hantavirus pulmonary syndrome real time RT PCR kit contains a specific ready to use system for the detection of Hantavirus pulmonary syndrome by Reverse Transcription Polymerase Chain Reaction RT PCR in the real time PCR system The master contains Super Mix for the specific amplification of hanta virus RNA The reaction is done in one step real time RT PCR The first step is a reverse transcription RT during which the hanta virus RNA is transcribed into cDNA Then a thermostable DNA polymerase is used to amplify the specifi
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